AIM: To study the effect of Buyang Huanwu Decoction (BYHWD) on the antioxidant enzymes and drug-metabolizing enzymes in rat liver. METHOD: Following treatment of rats with BYHWD at 6.42, 12.83, or 25.66 g·kg(-1) per day for 15 days, microsomes and cytosols isolated from the liver tissues were prepared by differential centrifugation according to standard procedures. The activities of the antioxidant enzymes and cytochrome b5, NADPH-cytochrome P450 reductase, CYP3A, CYP2E1, UGT, and GST of the rat livers were determined by UV-Vis spectrophotometer. RESULTS: The activities of ALT, AST, antioxidant enzymes, and the Hepatosomatic Index in serum were not significantly affected. In cytosols, the activity of CAT was significantly increased at the dosage of 12.83 g·kg(-1), and all the other antioxidant activities and MDA levels were not affected by this treatment. BYHWD had no effect on cytochrome b5, NADPH-cytochrome P450 reductase, CYP3A, and UGT. At the highest dose (25.66 g·kg(-1)), the activity of CYP2E1 was significantly inhibited, and the activities of GST and the level of GSH were increased. CONCLUSION BYHWD is safe for the liver, and has the functions of detoxification and antioxidant. Patients should be cautioned about the herb-drug interaction of BYHWD and CYP2E1 substrates.
Animals ; Antioxidants ; metabolism ; pharmacology ; Catalase ; metabolism ; Cytochrome P-450 CYP2E1 ; metabolism ; Cytosol ; Drugs, Chinese Herbal ; pharmacology ; Glutathione ; metabolism ; Glutathione Transferase ; metabolism ; Herb-Drug Interactions ; Inactivation, Metabolic ; drug effects ; Liver ; drug effects ; enzymology ; Male ; Microsomes ; Rats, Sprague-Dawley

OBJECTIVETo investigate the antiinflammatory activities of aqueous extract of Occimum gratissmium (OGE) with emphasis on expression of proinflammatory cytokines in Lipopolysaccharide (LPS)-stimulated epithelial cell BEAS-2B.

METHODSEffects of OGE on cell viability were determined by MTT assay. mRNA expression were analyzed by and reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Activation of kinase cascades was investigated by immunoblot. Intracellular reactive oxygen species (ROS) was analyzed by flow cytometry.

RESULTSOGE (<200 μg/mL) treatment or pretreatment and following LPS exposure slightly affected viability of BEAS-2B cells. Increase of interleukin (IL)-6 and IL-8 and the elevated level of intracellular ROS in LPS-stimulated BEAS-2B cells were diminished by OGE pretreatment in a dose-dependent manner. OGE suppressed inflammatory response-associated mitogen-activated protein kinases (MAPKs) and Akt activation. Additionally, OGE pretreatment increased level of cellular inhibitor of κBα (IκBα) and inhibited nuclear translocation of nuclear factor kappa B (NF-κB).

CONCLUSIONThese findings indicate that significant suppression of IL-6 and IL-8 expressions in LPS-stimulated BEAS-2B cells by OGE may be attributed to inhibiting activation of MAPKs and Akt and consequently suppressing nuclear translocation of NF-κB.

Cell Nucleus ; drug effects ; metabolism ; Cell Survival ; drug effects ; Cytosol ; drug effects ; metabolism ; Epithelial Cells ; drug effects ; enzymology ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; I-kappa B Proteins ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Interleukin-8 ; genetics ; metabolism ; Intracellular Space ; drug effects ; metabolism ; Lipopolysaccharides ; pharmacology ; Mitogen-Activated Protein Kinases ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Ocimum ; chemistry ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Protein Transport ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Respiratory System ; cytology ; Water

The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.
Animals ; Ascaris suum ; metabolism ; Calcineurin ; metabolism ; Calcium ; metabolism ; Calmodulin ; metabolism ; Cytoskeleton ; metabolism ; Cytosol ; metabolism ; Egtazic Acid ; analogs & derivatives ; pharmacology ; Helminth Proteins ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; metabolism ; Male ; Membrane Potential, Mitochondrial ; physiology ; Mitochondria ; metabolism ; Pseudopodia ; metabolism ; Signal Transduction ; Sperm Motility ; Spermatids ; drug effects ; physiology ; Spermatogenesis ; Type C Phospholipases ; metabolism

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans- Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.
Animals ; Brefeldin A ; pharmacology ; Cell Line, Tumor ; Cytosol ; drug effects ; metabolism ; Humans ; Intracellular Space ; drug effects ; metabolism ; Membrane Proteins ; metabolism ; Protein Transport ; drug effects ; Rats ; Vesicular Transport Proteins ; metabolism ; trans-Golgi Network ; drug effects ; metabolism

OBJECTIVETo investigate the involvement of apoptosis inducing factor (AIF) in caspase-independent pathway mediating apoptosis of cultured renal tubular epithelial cells induced by cisplatin (CP).

METHODSWestern Blot analysis and real-time PCR were performed to detect cytosol AIF (cAIF), nuclear AIF (nAIF) and AIF mRNA expression in cultured renal epithelial cells (HK-2) treated with cisplatin (CP) at various concentrations (0 - 200 micromol/L) and time courses (0 - 12 h). Immunofluorescence analysis was used to detect the AIF protein distribution in HK-2 cells. Pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA treatment, TUNEL and flow cytometer were used to measure the suppression of apoptosis induced by CP in HK-2 cells.

RESULTSThe expressions of cAIF, nAIF protein and AIF mRNA were all increased to some extent in HK-2 cells treated with CP at various concentrations and time points. cAIF expression was 2.3-fold (P < 0.05) increased after 25 micromol/L CP treatment for 12 h and 1.7-fold (P < 0.01) increased after 50 micromol/L CP treatment for 3 h, compared with that of control groups, and showed a concentration- and time-dependent increment. The nAIF expression reached a peak (4.3-fold increase) (P < 0.005) after 150 micromol/L CP treatment for 12 h and 3.7-fold incease (P < 0.05) after 50 micromol/L CP treatment for 9 h, compared with that of the 25 micromol/L group and 3 h group, respectively. The expression of nAIF was approximately consistent with cleaved-PARP expressive pattern. Real-time PCR showed that AIF mRNA increased gradually with prolonged treatment with 50 micromol/L CP and reached a peak at 9 h. Immunofluorescence assay showed AIF translocation from cytosol to nuclei in some cultured HK-2 cells treated with CP. Applying pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA to CP-treated HK-2 cells, the apoptotic rates were decreased by 60.1% and 39.2%, respectively. The inhibitory effect on HK-2 cell apoptosis was even more significant with combination of both Z-VAD-FMK and AIF-siRNA.

CONCLUSIONThe AIF activation and translocation to nuclei with the increment of its mRNA expression mediates CP-induced apoptosis of renal tubular epithelial cells in vitro. It may provide a new therapeutic target for protecting from nephrotoxciity of cisplatin.

Amino Acid Chloromethyl Ketones ; pharmacology ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Inducing Factor ; genetics ; metabolism ; Caspase Inhibitors ; Cell Nucleus ; metabolism ; Cells, Cultured ; Cisplatin ; administration & dosage ; pharmacology ; Cytosol ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Epithelial Cells ; cytology ; metabolism ; Humans ; Kidney Tubules ; cytology ; Protein Transport ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics

Endothelins (ETs), which were originally found to be potent vasoactive transmitters, were known to be implicated in nervous system, but the mode of mechanism remains unclear. ETs (ET-1, ET-2, and ET-3) were added to HN33 (mouse hippocampal neuron chi neuroblastoma) cells. Among the three types of ET, only ET-1 increased the intracellular calcium levels in a PLC dependent manner with the induction of ERK 1/2 activation. As the result of ET-1 exposure, the survival rate of HN33 cells and the PKCalpha translocation into the plasma membrane were increased. We suggest that ET-1 participated in the neuroprotective effect involving the calcium-PKCalpha-ERK1/2 pathway.
Animals ; Apoptosis/*drug effects ; Calcium/*metabolism ; Cell Line ; Cell Survival/drug effects ; Cytosol/drug effects/metabolism ; Endothelin-1/*pharmacology ; Endothelin-2/pharmacology ; Endothelin-3/pharmacology ; Estrenes/pharmacology ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Immunoblotting ; Mice ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Neurons/*cytology/drug effects/*enzymology ; Neuroprotective Agents/pharmacology ; Phosphoproteins/metabolism ; Protein Kinase C-alpha/*metabolism ; Protein Transport/drug effects ; Pyrrolidinones/pharmacology ; Serum

To investigate Bax translocation from cytosol into mitochondria induced by staurosporine (STS) in GFP-Bax-tagged MCF7 stable cell line, the viability was measured by MTT method. Bax translocation from cytosol into mitochondria was investigated under the fluorescence microscope. The dose-effect and time-course relationships were also observed and the percentage of GFP-Bax punctuate cells were calculated. Immunofluoresence method was used to observe Bax translocation to mitochondria, Cyt-c release from mitochondria and Annexin V label. The TMRE assay was used to investigate membrane pertential (Deltapsim) and function of mitochondria. Western blotting was used to observe the mechanism of apoptosis induced by STS. The results showed that STS can induce Bax translocation from cytoplasm to mitochondria, Cyt-c release from mitochondria and Annexin V label. The Western blotting analysis presented the inhibitory effect on apoptosis induced by STS of SP600125 which is a specific JNK inhibitor. The study revealed the mechanism of STS induced apoptosis associated with JNK activated pathway.
Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Cytosol ; metabolism ; Humans ; MAP Kinase Kinase 4 ; antagonists & inhibitors ; Membrane Potentials ; drug effects ; Mitochondria ; metabolism ; Protein Transport ; drug effects ; Staurosporine ; pharmacology ; bcl-2-Associated X Protein ; metabolism

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal thatgammagammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.
Anthracenes/pharmacology ; Apoptosis/drug effects/*genetics ; Carbazoles/pharmacology ; Cell Cycle/drug effects/genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/*biosynthesis/genetics ; Cytosol/drug effects/enzymology/ultrastructure ; DNA Damage/drug effects/genetics ; Doxorubicin/pharmacology ; Histones/*metabolism ; Humans ; Indole Alkaloids/pharmacology ; MAP Kinase Kinase 4/antagonists & inhibitors ; Nerve Growth Factor/antagonists & inhibitors/metabolism ; Phosphorylation/drug effects ; Protein Binding ; *Protein Transport/drug effects/genetics ; Receptor, trkA/antagonists & inhibitors/*genetics/metabolism ; Signal Transduction ; Transfection

To investigate the existence of nongenomic action of 17β-estradiol (E(2)) in the delayed implantation mouse endometrial stromal cells, the changes in intracellular calcium concentration ([Ca(2+)](i)) and the upstream of calcium signal in vitro were detected. The experiment was composed of two parts. Firstly, the change in [Ca(2+)](i) in endometrial stromal cells induced by E(2) under different conditions was detected. The mice were divided into 6 groups as follows: 0.1% dimethylsulfoxide (DMSO) control group, 1×10(-8) mol/L bovine serum albumin (BSA) control group, 1×10(-8) mol/L E(2) group, 1×10(-8) mol/L E(2) conjugated with BSA (E(2)-BSA) group, 1×10(-8) mol/L E(2) + calcium-free medium group, 1×10(-8) mol/L E(2) + 5 mg/mL tamoxifen group, with 4 mice in each group. The endometrial tissue was obtained from delayed implantation mice at pregnant day 7, and digested by incubation of tissue minces in Hankos balanced salts (HBSS, pH 7.2), which contained glucose (1 g/L), and collagenase I (0.125%), for 1 h at 37 degrees C. The stromal cells were preloaded with 2.5 mmol/L Fluo-3/AM, a fluorescent probe of calcium, for 30 min. A confocal laser scanning microscope, which fixed the wave length of excitation and emission at 488 nm and 526 nm, respectively, was used to detect the change in [Ca(2+)](i). Secondly, the mechanism of E(2) effects in endometrial stromal cells was investigated. Immunofluorescent method was used to detect the change in phosphorylation of phospholipase C (PLC) before and after the stromal cells were treated with E(2) for 5 min, 15 min, and 30 min. Seven delayed implantation mice were used. The results were as follows. [Ca(2+)](i) increased immediately and reached the maximum at 15 min after the stromal cells were treated with 1×10(-8) mol/L E(2) and returned to the normal level at 30 min. In E(2)-BSA group and E(2) + calcium-free medium group the same results were obtained as that in E(2) group, but there was no increase of [Ca(2+)](i) in DMSO and BSA groups. Tamoxifen, a traditional antagonist of estrogen receptor, did not inhibit the increase in [Ca(2+)](i) induced by E(2). Immunofluorescent results showed that the change in phosphorylated-PLC level had the same trend as [Ca(2+)](i) after the cells were treated with E(2). Compared with that in the control group, the immunofluorescent intensity increased at the beginning and achieved the maximum at 15 min (P<0.001), then declined to the normal level at 30 min. These results suggest that the rapid response of [Ca(2+)](i) induced by E(2) in the endometrial stromal cells in delayed implantation mice is possibly carried out through a nongenomic pathway, and the transmembrane signal transduction is related to the phosphorylation of PLC in this process.
Animals ; Calcium ; chemistry ; Culture Media ; Cytosol ; chemistry ; Endometrium ; cytology ; Estradiol ; pharmacology ; Female ; Mice ; Phosphorylation ; Pregnancy ; Receptors, Estrogen ; Signal Transduction ; Stromal Cells ; cytology ; drug effects ; Tamoxifen

Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities.
Amino Acid Chloromethyl Ketones ; pharmacology ; Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Caspase Inhibitors ; Caspases ; metabolism ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Cytosol ; drug effects ; metabolism ; Enediynes ; pharmacology ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; biosynthesis ; genetics